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First published online January 12, 2006
doi: 10.1242/10.1242/dev.02238
Review |
1 Faculty of Life Sciences, The University of Manchester, Oxford Road,
Manchester M13 9PT, UK.
2 Developmental Neurobiology, National Institute for Medical Research, Mill
Hill, London NW7 1AA, UK.
Authors for correspondence (e-mail: hilary.ashe{at}manchester.ac.uk; james.briscoe{at}nimr.mrc.ac.uk)
SUMMARY
Morphogens act as graded positional cues that control cell fate specification in many developing tissues. This concept, in which a signalling gradient regulates differential gene expression in a concentration-dependent manner, provides a basis for understanding many patterning processes. It also raises several mechanistic issues, such as how responding cells perceive and interpret the concentration-dependent information provided by a morphogen to generate precise patterns of gene expression and cell differentiation in developing tissues. Here, we review recent work on the molecular features of morphogen signalling that facilitate the interpretation of graded signals and attempt to identify some emerging common principles.
Introduction
The transformation of the spatial distribution of naïve cells in a
developing tissue into an organised arrangement of cell differentiation is
fundamental to the development of multicellular organisms. More than a century
ago, evidence began to accumulate that cells receive `positional information'
that instructs them to develop in specific ways, depending on their location
within a tissue (Wolpert,
1996
). Over the intervening decades, the potential for signalling
gradients to provide this positional information has become a
much-investigated and -debated subject, and the term `morphogen' has been
coined to describe such signals. Today the morphogen concept continues to form
the basis of many models of pattern formation
(Lewis et al., 1977;
Green and Smith, 1991;
Gurdon and Bourillot, 2001;
Tabata and Takei, 2004).
Typically, in current models it is proposed that a signal produced from a
defined localised source forms a concentration gradient as it spreads through
surrounding tissue (Fig. 1A).
The graded signal then acts directly on cells, in a concentration-dependent
manner, to specify gene expression changes and cell fate selection. Thus, the
concentration of ligand provides cells with a measure of their position
relative to the source of the signal and organises the pattern of cell
differentiation. Experimental evidence from tissues in both vertebrates and
invertebrates indicates that several molecules appear to function as graded
signals. The roles of these signals range from the establishment of the
initial polarities of embryos to specification of cell identity in specific
tissues, notably limb appendages and the nervous system in both vertebrates
and Drosophila. The examples we focus on in this review are
introduced in Fig. 1. Evidence
in support of these signals acting as graded morphogens has been summarised in
recent reviews (Gurdon and Bourillot,
2001; Tabata and Takei,
2004).
Although the morphogen concept has provided an enduring and valid framework
for understanding pattern formation, it raises many mechanistic issues. Much
attention has focused on how the distribution of a morphogen through a tissue
establishes and maintains a gradient of activity
(Vincent and Dubois, 2002;
Tabata and Takei, 2004);
however, how the signal is perceived and interpreted in a graded manner by the
receiving cells has received less consideration. Nonetheless, this represents
an equally important element of the morphogen hypothesis. Crucial to
understanding the mechanism of morphogen activity is determining how a graded
signal is transformed into alterations in gene expression programmes, such
that the positional information supplied by the morphogen produces the
appropriate spatial pattern of cellular differentiation. To understand how
this is accomplished, several questions have to be addressed. How does the
signal transduction pathway transmit graded information intracellularly to
control concentration-dependent differential gene expression? How is a
continuous gradient transformed into discrete changes in gene expression that
ultimately determine the choice of cell fate from the available alternatives?
And how does graded signalling accommodate fluctuations in biological
conditions to achieve the necessary robustness required for accurate
developmental patterning? By focusing on specific examples, we review recent
work that addresses these questions and, where possible, we highlight some of
the general principles that appear to be shared between different morphogen
gradients.
Morphogen signal transduction pathways are linear and transmit graded information
How many thresholds does a morphogen control?
At a minimum, to meet the definition of a morphogen, a graded signal must
be able to direct the generation of at least two distinct cell types at
different concentrations. Theoretical analysis has raised the possibility that
graded signals can achieve up to 30 thresholds
(Lewis et al., 1977); however,
empirical evidence has typically identified between three and seven distinct
thresholds. For example, the Dorsal (Dl) gradient appears to specify at least
four, and as many as seven, distinct thresholds of gene expression along the
dorsoventral (DV) axis of Drosophila embryos
(Stathopoulos and Levine,
2002a). A concentration gradient of activin is able to induce five
cell states in Xenopus blastula cells
(Green et al., 1992), and a
similar number of neuronal subtypes appears to be produced by graded Sonic
Hedgehog (Shh) signalling in the neural tube
(Ericson et al., 1997;
Pierani et al., 1999). In each
of these cases, additional signals are believed to promote or cooperate in the
forming of some of the threshold responses, so whether a single morphogen
acting alone produces each of the observed threshold responses remains
unknown. In other well-studied cases, fewer defined thresholds have been
clearly identified, for example Wingless (Wg) signalling in the
Drosophila wing imaginal disc promotes three thresholds of gene
expression (Tabata and Takei,
2004), whereas graded Decapentaplegic (Dpp) signalling is
responsible for at least three threshold responses in Drosophila
embryos and the wing disc (Ashe et al.,
2000; Affolter et al.,
2001).
Small morphogen concentration changes are sensed
In the case of the vertebrate morphogens activin, bone morphogenetic
protein (Bmp) 4 and Shh, the dose responses of cells have been assayed
(Green et al., 1992;
Wilson et al., 1997;
Ericson et al., 1997). For
activin and Shh, the full range of responses is elicited over a 25- to 50-fold
concentration range with relatively small, two- to threefold, changes in
concentration being sufficient to switch cells between alternative fates.
Moreover, for other graded signals, the evidence also suggests that
comparatively moderate changes in signalling strength are sufficient to alter
significantly the response of cells. For example, a gradient of Dpp regulates
the dose-dependent expression of target genes in dorsal regions of
Drosophila embryos. Altering the gene dose of dpp by
decreasing the gene copy number to one or by increasing it to three or four
has significant effects on the position at which target genes are expressed
(Ashe et al., 2000). Consistent
with this, the injection of dpp transcripts into Drosophila
embryos is sufficient to promote the development of ectodermal cells with
incremental two- to fourfold increases in the injected concentration eliciting
progressively more-dorsal cell fates
(Ferguson and Anderson,
1992).
|
). Moreover, the transmembrane protein Smoothened is
necessary and sufficient to induce the multiple neuronal subtypes
characteristic of Shh signalling in the vertebrate ventral neural tube
(Hynes et al., 2000;
Wijgerde et al., 2002). In the
case of Bmp signalling in the Drosophila dorsal ectoderm, the
presence of Dpp and a second Bmp-type ligand, Screw, facilitates the
activation of different receptor complexes that contain distinct combinations
of receptors molecules. Despite this, within the cell the same transcriptional
effectors transduce signals emanating from each receptor complex
(Shimmi et al., 2005).
Employing a single species of receptor to transmit concentration-dependent
information does not preclude the downstream induction of different branches
of a signalling pathway at different ligand concentrations. For example, in
tissue-culture models, a different set of proteins appears to be
phosphorylated and activated at low concentrations of platelet-derived growth
factor compared with high concentrations
(Rankin and Rozengurt, 1994).
However, this type of mechanism does not appear to be favoured in the
perception of morphogen gradients. Instead, linear signalling pathways seem to
be the rule. An elegant demonstration of this comes from the ability of a Dl
nuclear gradient to pattern the DV axis of the early Drosophila
embryo. In the embryo, graded activation of the Toll transmembrane receptor by
Spatzle leads to the induction of Pelle kinase, and ultimately nuclear
translocation of Dl (Fig. 1C)
(Stathopoulos and Levine,
2002a). Ectopic gradients of either Toll or Pelle can provide
positional information and control multiple patterning thresholds, providing
evidence of a linear signalling pathway in which differences in the number of
activated Toll receptors are transduced to a gradient of Pelle activity that,
in turn, establishes a gradient of nuclear Dl
(Stathopoulos and Levine,
2002b). Studies of other morphogen signalling pathways indicate
that each pathway culminates in the post-translational regulation of the
activity of a single transcription factor or family of related transcription
factors that have overlapping functions. For example, a constitutively active
form of ß-catenin/Armadillo (Arm), the transcriptional mediator of Wg
signalling, is sufficient to induce the expression of both short range and
long targets of Wg signalling in the Drosophila wing disc
(Zecca et al., 1996).
Similarly, Smad1 and Smad2, the mediators of Bmp4 and activin signalling,
respectively, are sufficient to transduce the graded responses to these
signals (Wilson et al., 1997;
Shimizu and Gurdon, 1999).
Active versions of the Gli3 protein, the Shh transcriptional mediator (see
Fig. 1E), can recapitulate the
patterning activity of Shh in the chick neural tube
(Stamataki et al., 2005).
Morphogen concentration determines the level of transcriptional effector activated
The apparent lack of signalling pathway branching, together with the
sufficiency of single transcriptional effectors to mediate the full range of
responses to a morphogen, indicate that changes in the extracellular morphogen
concentration should be reflected directly by differences in the activity of
the relevant transcriptional effectors. In general, this appears to be the
case. For example, the threefold difference in activin concentration that
causes a switch in gene expression in Xenopus cells is mimicked by a
comparable change in the level of nuclear Smad2
(Shimizu and Gurdon, 1999).
Similarly, the graded activity of Bmp4 can be recapitulated with corresponding
concentration changes in ectopic Smad1
(Wilson et al., 1997).
Moreover, the incremental two- to threefold changes in Shh concentration,
which determine alternative neuronal subtypes, are mimicked by equivalent
small changes in Gli activity levels, indicating that a gradient of Gli
activity reflects graded Shh signalling
(Stamataki et al., 2005).
Together, these observations suggest that morphogen gradient interpretation
requires target genes to be able to interpret two- to threefold changes in
transcriptional effector in order to generate distinct transcriptional
responses. Consistent with this, the Bicoid (Bcd) target gene
hunchback (hb) appears able to discriminate between
approximately twofold changes in Bcd concentration
(Struhl et al., 1989).
In other cases, it is less clear if there is a direct correlation between
changes in the extracellular ligand concentration and changes in
transcriptional strength. For example, although the graded activation of
Mothers against Dpp (Mad), the transcriptional effector of the Dpp pathway
(see Fig. 1D), depends on its
ligand, studies in the Drosophila wing disc have revealed that a
sudden transition in the level of activated Mad occurs that does not coincide
with a similar, abrupt change in the distribution of Dpp
(Teleman and Cohen, 2000). It
is possible that this is because the Dpp visualised in these experiments does
not accurately reveal the distribution of Dpp that is able to engage and
activate its receptors. Alternatively, the deviation may be due to the
modulation of the activated Mad profile by additional factors, such as
Daughters Against Dpp (Dad), which is an inhibitory Smad, the Saxophone
receptor, the levels of the SARA adaptor protein or the Smurf ubiquitin ligase
(Teleman and Cohen, 2000). For
other graded signals, the quantitative relationship between ligand
concentration, pathway activity and transcriptional effectors remains to be
determined.
The linearity of signalling pathways implies that the signal transduction
machinery transmits concentration-dependent information with sufficient
fidelity to mediate differential responses. Consequently, changes in ligand
concentration control proportionate quantitative changes in the activity of
the transcriptional effectors. This provides a contrast to those signalling
pathways that display bistable or ultra-sensitive responses that confer
monotonic, switch-like responses (Monod
and Jacob, 1961; Ferrell,
2002). A well-studied example of this type of response is the
maturation of oocytes, which is induced at a crucial threshold of progesterone
signalling through the activation of a mitogen-activated protein kinase (Mapk)
cascade (Ferrell and Machleder,
1998). Similar switch-like behaviour may be elicited by other
signals, such as some receptor tyrosine kinase receptor pathways that activate
the Ras-Mapk pathway. Although this type of binary switching behaviour is
relevant in some biological settings, it does not allow the transduction of a
graded signal responsible for controlling multiple cell fate decisions at
different concentration thresholds. This raises the possibility that the
molecular mechanisms of signalling pathways capable of transmitting
concentration-dependent responses are distinct from those that provide simple
monotonic responses. Addressing this issue will require a detailed analysis
and comparison of the mechanisms of signal transduction and of the strategies
employed to transfer graded information accurately from receptor to the
nucleus.
Strategies employed in the regulation of differentially responsive genes
The mechanisms of gene regulation by morphogen signalling must provide a
means to translate small differences in signal strength into threshold
responses in which all-or-none changes in gene expression allow the selection
of discrete cell identities in the developing tissue. More than a generation
ago, strategies that could explain this phenomenon were proposed
(Monod and Jacob, 1961), and
some of these ideas are beginning to re-emerge from more recent molecular
studies. We attempt to categorise these strategies into general design
features that can account for differential gene regulation by graded
signalling (Fig. 2). Clearly,
there are overlaps between these categories and the list is not exhaustive. It
is apparent that most, if not all, of the well-studied morphogen pathways use
a combination of these mechanisms to control target gene expression. To
illustrate the key features of each of the strategies, we have outlined
examples of their use in the interpretation of specific morphogen
gradients.
Binding-site affinity
A major mechanism that has been extensively investigated exploits
differences in the affinity of the transcriptional effector for binding to
sites with different DNA sequences (Fig.
2A). A paradigm for this is the Dl gradient in the early
Drosophila embryo, which directs DV patterning and gastrulation
through the concentration-dependent activation and repression of target genes
(Stathopoulos and Levine,
2004). Extensive studies of specific enhancers that respond to
different thresholds of Dl have revealed a detailed picture of the mechanism
of gene regulation. Based on their responsiveness to Dl, target genes have
been classified into different categories. Type I genes, such as
twist (twi) are activated in the presumptive mesoderm where
there are peak levels of nuclear Dl (Fig.
1C). The enhancers of these genes tend to have low-affinity
Dl-binding sites that are only occupied at the highest Dl concentration, thus
limiting the expression of type I genes to the presumptive mesoderm
(Jiang and Levine, 1993). By
comparison, enhancers of type II genes, such as rhomboid
(Fig. 1C), contain
high-affinity Dl-binding sites that are bound and activated by the lower
levels of Dl that are present in the ventral neuroectoderm
(Ip et al., 1992a). Recent
computational analysis of a large set of Dl-responsive enhancers from the
genomes of D. melanogaster and related species has confirmed that Dl
affinity is a major determinant of the expression domains of Dl target genes
(Papatsenko and Levine, 2005).
However, a high-affinity Dl-binding site does not necessarily lead to the
activation of transcription when Dl is present. In some cases, Dl bound to a
high-affinity site can also repress transcription, indicating that enhancer
architecture also plays a significant role in determining the responsiveness
of genes to Dl (Stathopoulos and Levine,
2004). Moreover, cooperative interactions between Dl and other
factors also significantly influence the responsiveness of some genes.
A second example is interpretation of the Bcd gradient, which is
responsible for regulating gene activity along the anteroposterior (AP) axis
in the Drosophila embryo. Early studies of Bcd interpretation
identified the affinity of Bcd-binding sites as a key determinant for setting
the limits of expression of the hb target gene
(Fig. 1B). Decreasing Bcd
affinity leads to a more anterior restricted expression pattern where Bcd
levels are higher. Thus, a model was proposed for the interpretation of the
Bcd gradient, in which genes with anterior restricted expression have
low-affinity Bcd-binding sites in their enhancer and consequently require a
high Bcd concentration for occupancy and activation. Conversely, the
higher-affinity sites in the hb enhancer allow expression at more
posterior positions where the Bcd concentration is lower
(Driever et al., 1989;
Struhl et al., 1989). In
support of this model, the orthodenticle gene, which is regulated by
a low Bcd affinity enhancer, has a narrow expression pattern
(Gao et al., 1996)
(Fig. 1B).
It is not only in the precellular embryo that the response of genes to
graded transcription factor activation uses binding-site affinity. This
mechanism is also relevant in more conventional settings post cellularisation,
such as the interpretation of the extracellular gradient of Dpp in the
Drosophila embryo. In response to peak levels of Dpp signalling at
the dorsal midline of the embryo, the target gene Race is expressed
in a narrow stripe of cells in the presumptive amnioserosa
(Fig. 1D). The enhancer
responsible for this activity contains low-affinity binding sites for Mad, the
transcriptional effector of Dpp. Altering these sites to increase their
affinity for Mad broadens the associated expression pattern to that
characteristic of genes that are responsive to a lower threshold of Dpp
signalling (Wharton et al.,
2004).
Combinatorial inputs
Binding-site affinity can account for some of the morphogen gradient
readouts; however, in general, affinity alone is insufficient to direct the
full complement of transcriptional responses. For example, although the
affinity of Bcd-binding sites sets the limits of expression of the hb
target gene (Driever et al.,
1989; Struhl et al.,
1989), a computational study of a larger sample size of Bcd
cis-regulatory modules indicates that for most there is a poor correlation
between the strength of Bcd-binding clusters and the expression limits of a
gene. Moreover, only a few target genes appear to be activated by Bcd alone,
and the expression of these genes is restricted to the most anterior parts of
the embryo that contain peak Bcd levels
(Ochoa-Espinosa et al., 2005),
as observed for a synthetic reporter containing only Bcd-binding sites
(Crauk and Dostatni, 2005).
For many genes, the major determinant for the interpretation of positional
information is not the absolute Bcd affinity. Instead, other elements in
target gene promoters and the integration of positive and negative
transcriptional inputs from proteins bound to these elements can determine the
interpretation of the Bcd gradient. For genes activated in the middle and
posterior regions of the embryo, most enhancers of Bcd target genes tend to
have additional inputs from the Hb, Caudal (Cad) and/or Krüppel (Kr)
transcription factors (Ochoa-Espinosa et
al., 2005). Hb and Cad are maternally expressed and zygotically
activated and repressed by Bcd at the transcriptional and translational
levels, respectively (Driever and
Nusslein-Volhard, 1989; Dubnau
and Struhl, 1996; Rivera-Pomar
et al., 1996). Both Hb and Cad augment Bcd-dependent
transcriptional activation (La Rosee et
al., 1997; Simpson-Brose et
al., 1994). Therefore, the Bcd gradient may function with Hb
and/or Cad to establish a broad domain where enhancer activation can occur,
and the balance of positive and/or negative inputs from these and other
transcription factors would determine the limits of an expression domain
(Ochoa-Espinosa et al., 2005).
The transcriptional repressor Kr may be one such negative input that sets a
sharp posterior border of some Bcd targets
(Kraut and Levine, 1991). As
well as binding sites for other transcriptional effectors, the arrangement of
Bcd-binding sites also influences gene expression, and the data indicate that
Bcd binds cooperatively to DNA. Therefore, Bcd binding to a high-affinity site
potentiates binding to an adjacent low-affinity site
(Burz et al., 1998). Expression
of a Bcd protein with a mutation that disrupts cooperativity in the embryo
leads to an anterior shift in the expression patterns of target genes, such as
hb, and reduced sharpness of their posterior borders
(Lebrecht et al., 2005).
|
)
has revealed that, just as type I threshold genes tend to have lower Dl
affinity than do type II genes, there is a similar trend in these promoters
for the affinity of another transcription factor, Twi. Moreover, type II
thresholds tend to have a fixed orientation of, and spacing between, Dl and
Twi sites. This is consistent with the occurrence of synergistic interactions
between Dl and Twi transcription factors being important for the activation of
type II targets in the neurectoderm where Dl and Twi levels are low
(Papatsenko and Levine, 2005).
Type II enhancers commonly have an additional positive input from the
Suppressor of Hairless [Su(H)] activator
(Erives and Levine, 2004) and
a negative input from the Snail repressor. snail is a Dl target gene
that is activated in the presumptive mesoderm
(Ip et al., 1992b), thereby
excluding expression of type II genes from the mesoderm
(Kosman et al., 1991). In
contrast to type II enhancers, there is a negative correlation between the
quality of Dl and Twi sites at type I enhancers, i.e. a good Dl site is
associated with a poor Twi site, and vice versa. This suggests that, at those
enhancers where there are peak levels of Dl and Twi, the activators function
in a compensatory manner (Papatsenko and
Levine, 2005). Importantly, studies with synthetic enhancers also
indicate that the presence of Twi and Dl sites leads to a sharper expression
pattern, in comparison with the weaker, fuzzy pattern that is observed with Dl
alone (Szymanski and Levine,
1995). Thus, it is clear that in addition to the affinity of
binding sites for the main transcriptional effector of a morphogen, the
integration of positive and negative inputs onto an enhancer is an important
determinant of threshold responses (Fig.
2B).
Feed-forward loops
The inclusion of combinatorial inputs into the control of differential gene
expression allows complex regulatory relationships to develop between
responding genes. One such relationship is the feed-forward loop
(Fig. 2C), an example of which
has recently been described for the activation of Race by Dpp
signalling. In addition to the affinity of Mad-binding sites in the
Race enhancer (Wharton et al.,
2004), the transcription factor Zerknüllt (Zen) plays a
crucial role in Race activation. Zen and Mad bind to adjacent sites
in the Race enhancer, and a direct interaction between them is
necessary for Race activation (Xu
et al., 2005). zen is itself a Dpp-regulated gene that
depends on peak levels of Dpp signalling
(Rushlow et al., 2001). Thus,
for Race to be induced, peak levels of Dpp signalling need to
activate high levels of Mad and to induce Zen expression, which function
together to activate Race (Xu et
al., 2005). This type of regulatory genetic network, in which
transcription factor X activates transcription factor Y, and together X and Y
activate target Z, is termed a feed-forward loop
(Lee et al., 2002).
The Mad-Zen feed-forward loop may represent a general strategy that is used
to activate other peak Dpp target genes
(Xu et al., 2005). It is
certainly the case that feed-forward loops operate in other
morphogen-responsive gene networks. For example, Twi, which functions with Dl
to regulate genes along the DV axis, is itself encoded by a Dl-responsive gene
(Jiang and Levine, 1993). The
recurrence of feed-forward loops in the interpretation of early
Drosophila embryo morphogen gradients suggests that his type of
regulatory circuit is particularly suitable for gradient interpretation. Data
from other systems reveal that feed-forward loops are useful for
discriminating between erratic external signals to ensure that activation only
occurs in response to persistent signalling, thus providing a means to buffer
against small fluctuations in signal
(Shen-Orr et al., 2002).
Moreover, the co-incidence requirement inherent in feed-forward loops can also
provide highly sensitive responses to small changes in signal level
(Goldbeter and Koshland,
1984), a feature that would allow threshold responses to be
generated in response to small changes in the initial signalling strength.
Positive feedback
The autoregulation of, or positive-feedback loops (see
Fig. 2D) in, responding genes
can also play a role in gradient interpretation and provide a mechanism for
the generation of all or none responses at threshold levels of signalling. A
well-characterised example of this is the regulation of Hoxb4 in the
vertebrate hindbrain (Gould et al.,
1998; Gould et al.,
1997). A gradient of retinoic acid (RA) confers positional
information along the AP axis of the forming vertebrate hindbrain and is
responsible for determining the anterior limit of the induction of
Hoxb4. RA activates nuclear RA receptors (RARs), and these receptors
bind to a defined enhancer region in the Hoxb4 locus to activate its
expression. At early stages of hindbrain development, this mechanism
establishes a diffuse anterior expression border of Hoxb4. A second enhancer
element, the late enhancer element, within the Hoxb4 locus is
responsive to Hoxb4 protein itself. Therefore, at later developmental stages,
following RA-mediated induction of Hoxb4, this element responds to
the induced Hoxb4 and is sufficient to direct expression of this gene up to
the normal anterior boundary of gene expression. Thus, graded RA activity
initiates Hoxb4 expression, Hoxb4-mediated autoregulation by
Hoxb4 refines and maintains its expression as hindbrain development
progresses. Hoxb4 regulates RARß in a similar manner, indicating that a
reciprocal positive feedback circuit exists between these proteins that
generates and maintains the discrete boundaries of Hoxb4 expression
(Serpente et al., 2005).
Cross repression
Repressive interactions between morphogen-regulated genes are also
important for gradient interpretation (Fig.
2E). A well-studied example is the contribution of cross
repression to the partition of the Drosophila neuroectoderm into
three columns along the DV axis (Cowden
and Levine, 2003). This subdivision is mediated by three homeobox
transcription factors (Vnd, Ind and Msh) that demarcate the ventral,
intermediate and dorsal columns, respectively. Distinct thresholds of Dl
signalling induce these genes, but the production of the distinct columns of
gene expression, which are delimited by abrupt switches in the expression of
each homeodomain protein, depends on asymmetric cross-regulatory interactions
between these proteins. In this way, the homeodomain proteins expressed in the
more ventral domains repress those expressed more dorsally. Thus, incremental
increases in Dl signalling result in the sequential activation of each gene
and in the corresponding repression of the genes induced by lower levels of Dl
activity - a process that has been termed `ventral dominance'.
The vertebrate nervous system displays a variation on this regulatory motif
that involves the use of mutual cross-repression, or reciprocal negative
feedback, between pairs of genes. Cells in the vertebrate neural tube respond
to graded Shh signalling by regulating the expression of a series of
transcription factors that include the homeodomain orthologues of Vnd, Ind and
Msh (Briscoe and Ericson,
2001). On the basis of their mode of regulation by Shh signalling,
these transcription factors are divided into two groups, termed class I and II
proteins. The expression of each class I protein is extinguished at distinct
thresholds of Shh activity; conversely, expression of the class II proteins
depends on Shh signalling. In vivo, the expression patterns of these genes
divides the ventral neural tube into sharply demarcated domains reminiscent of
those seen in Drosophila, with the ventral limit of most class I
proteins corresponding to the dorsal limit of expression of a class II
protein. This is achieved by selective cross-repressive interactions between
the complementary pairs of class I and class II proteins expressed in adjacent
abutting domains (Briscoe et al.,
2001; Briscoe et al.,
2000). In both the vertebrate and Drosophila nervous
system (Cowden and Levine,
2003), the repressive interactions establish gene-response
thresholds and generate the sharp boundaries of gene expression that ensure
each progenitor domain expresses a distinct set of transcription factors. This
mechanism converts a gradient of positional information into discrete
all-or-none changes in gene expression.
The principle of cross-regulatory interactions is also observed in other
developing tissues, indicating that it may represent a general strategy
deployed to interpret graded positional information. The Bcd gradient
specifies the expression domains of the Gap genes, which position the
downstream pair-rule and segment polarity genes necessary for segmentation of
the embryo (Jäckle et al.,
1986; Kraut and Levine,
1991). Both asymmetric and reciprocal repressive interactions
between Gap genes appear to form an intricate circuit. Strong reciprocal
repression between pairs of genes ensures mutual exclusivity of expression,
while asymmetric repression of anterior Gap genes by more posterior genes
leads to an anterior shift in their posterior boundaries
(Jaeger et al., 2004;
Monk, 2004)
(Fig. 2E). These findings
highlight a dynamic feature of Bcd gradient interpretation, whereby spatial
domains of gene expression can be repositioned by subsequent asymmetric
repressive interactions between Gap genes.
Reciprocal repressor gradient
A common feature of many morphogen gradients is the establishment of an
inverse gradient of a transcriptional repressor that is reciprocal to the
transcriptional effector activated by the signal
(Fig. 2A). In the case of Shh
and Wnt signalling, the primary transcriptional effectors of these pathways
mediate transcriptional repression in the absence of signalling, but are
converted to transcriptional activators upon signalling
(Giles et al., 2003;
Jacob and Briscoe, 2003). The
net effect of signalling, then, is formation of a gradient of transcriptional
activator with an opposing repressor gradient, a strategy that could augment
changes in transcriptional activity mediated by the morphogen. A variation in
this strategy is employed in the interpretation of the Dpp gradient in the
Drosophila wing imaginal disc. Here, the main role of Dpp signalling
appears to be the creation of a reciprocal gradient of the Brinker (Brk)
repressor protein. Mad and Medea directly repress Brk in a complex with the
Schnurri transcription factor (Pyrowolakis
et al., 2004), and sensitivity to Brk repression sets the
expression limits of the Dpp threshold responses, including spalt
(sal) and optomotor-blind (omb)
(Muller et al., 2003).
omb expression is repressed in mad mutant clones because of
the derepression of Brk. However, in brk mad double mutant clones
there is ectopic activation of omb, indicating that, for omb
expression, the only requirement for Dpp signalling is to repress Brk. By
contrast, expression of maximal levels of sal does require a positive
input from the Smads (Affolter et al.,
2001; Barrio and de Celis,
2004). In other developmental contexts, Mad and Brk have been
found to compete for the same binding sites
(Affolter et al., 2001),
although the relevance of this to the establishment of the wing target gene
expression domains is unclear.
How are interpretation strategies influenced by properties of the morphogen gradient?
It is apparent that, in each case, interpretation of morphogen signalling involves a combination of different mechanisms; it is difficult to deduce with certainty why one strategy is employed over another. However, in some cases, clues about this may come from specific features of the gradients themselves.
Interpretation of a step gradient
Although the standard view of a morphogen gradient is a continuous
gradient, the embryonic Dpp gradient is unusual in that it contains a
threshold, or step, in its distribution at the dorsal midline
(Ashe, 2005). This step is
mirrored by a similar plateau of high nuclear Smad concentration, which is
flanked by a shoulder of lower concentration
(Raftery and Sutherland,
2003). This unusual gradient distribution may help to generate
sharp borders of the peak and intermediate Dpp threshold responses, which
coincide with the stepped Smad nuclear gradient. In fact, the step gradient
may have obviated the need for a repressor to assist in the specification of
these Dpp threshold responses. Although sharp borders of threshold responses
tend to involve an additional repressor input, such as those described in the
patterning of the Drosophila and vertebrate nervous systems
(Cowden and Levine, 2003;
Briscoe and Ericson, 2001),
based on current knowledge, the establishment of the peak Dpp threshold
Race requires only inputs from activators (Zen and Smads)
(Wharton et al., 2004;
Xu et al., 2005).
Temporal integration
The interpretation strategy and regulatory circuit may also govern the
temporal response of cells to ongoing morphogen signalling. A striking
correlation between the strength and duration of signalling and the spatial
distribution of induced genes has been observed in a number of experiments.
For example, at a low concentration or short duration, activin signalling
induces Xbra at short range in Xenopus blastula cells, whereas with
increasing time or activin concentration, the field of Xbra-expressing cells
appears to move away from the source of signal
(Gurdon et al., 1995).
Likewise, studies of the relationship between time and concentration of Shh
signalling and the induction of different digits in the vertebrate limb
indicate that increasing the duration of Shh signalling results in the
generation of increasingly posterior digits
(Harfe et al., 2004;
Yang et al., 1997). One
possibility that would account for the temporal integration of morphogen
signalling is that signal duration is sensed by cells in a similar manner to
increasing signal strength - more signal results in the activation of
increasing amounts of the transcriptional effector. Alternatively it is
possible that the concentration and duration of signalling are not directly
equivalent. The identification of feed-forward loops and cross-regulatory
networks downstream of graded signals could offer an explanation. Accordingly,
the regulatory interactions between morphogen target genes would result in a
sequential induction of genes, providing a mechanism to explain changes in the
temporal response of cells to morphogens.
It is possible that the regulatory circuits also provide an explanation for
the hysteretic, or persistent, feature of the response of cells to a gradient
(Lewis et al., 1977). This
attribute, which has also been termed the `ratchet effect'
(Gurdon et al., 1995), results
in cells retaining gene expression profiles characteristic of the highest
concentration of signal to which they have been exposed. The induction of a
gene in a positive-feedback loop becomes self sustaining, while
cross-repression allows the persistence of the expressed gene and the
inhibition of its negative regulator. The prolonged maintenance of gene
expression profiles after a gradient has dissipated could relieve a
requirement for a long-lasting signalling gradient to be established and could
allow the consolidation of the positional identity of a cell.
Robustness and correction mechanisms in the interpretation of gradients
Quantitative aspects of morphogen activity appear at odds with normal
biological processes. Small changes in the concentration of an extracellular
signalling molecule can have dramatic consequences on cell fate, yet embryonic
development is able to cope with stochiometric fluctuations in gene expression
and with changes in environmental and genetic conditions, such as changes in
temperature and gene dose. Investigations of the mechanisms that underlie the
precision and robustness of different signalling pathways have largely focused
on morphogen distribution and the regulation of the signal transduction. Such
studies have found that a number of mechanisms appear to operate to increase
the reliability of graded signalling
(Eldar et al., 2004;
Freeman, 2000). In addition,
gene regulation strategies, such as feed-forward and positive-feedback loops,
may also contribute to the reliability of gene expression in response to
morphogen signalling. Moreover, it is apparent that several mechanisms also
exist to correct and refine initial morphogen patterning
(Box 1), which facilitate the
elimination (Namba et al.,
1997), rearrangement (Wijgerde
et al., 2002) or respecification
(Standley et al., 2001) of
mislocated cells. Understanding the molecular basis of these mechanisms and
analysing the contribution they make to the precise and reliable patterns
generated by morphogens requires considerable additional work.
Future perspectives
Much progress has been made towards identifying and understanding morphogen
gradients. Emerging from these studies are a number of principles and shared
strategies that we have attempted to outline in this review. Questions
relating to the molecular mechanisms of morphogen activity still need to be
addressed. One challenge is to understand how quantitative information is
faithfully transferred through signalling pathways. The realisation of this
goal depends on the development of reagents and techniques that will allow
live in vivo assays to be performed at the single-cell level. In most cases,
mechanistic inferences about how the quantitative differences in the
activation of genes are interpreted have been drawn from a limited number of
target genes, so it is unclear how general the conclusions are. However, with
the advent of computational approaches to enhancer identification
(Vavouri and Elgar, 2005) and
of ChIP-chip technology (Taverner et al.,
2004), it will be possible to address interpretation on a
genome-wide scale. In this way, the relative contributions of different
transcription factors throughout the duration of signalling can be crucially
assessed and incorporated into network solutions for gradient interpretation
(Stathopoulos and Levine,
2005). Moreover, the relationships between the regulatory motifs
deployed in the regulation of different genes need to be compared and placed
in the context of the entire genetic network controlled by a morphogen. How
the specific gene expression programmes then specify different cell fates also
needs to be determined, and progress has been made in this area with respect
to gastrulation of Drosophila embryos in response to the early Dl
gradient (Stathopoulos and Levine,
2004) and to the control of neuronal subtype identity in the
vertebrate neural tube (Lee and Pfaff,
2001). Again, whole-genome expression profiling can potentially
identify all in vivo morphogen targets from which a framework for cell fate
specification can be generated
(Stathopoulos and Levine,
2004). Details on the precision and refinement of gradient
interpretation are still vague in most cases, yet this is an important issue
for reliable embryonic development in the real-world conditions that are
experienced by most embryos that develop outside of a cosseted laboratory
environment. Coupled with this is the issue of how interpretation can remain
accurate when gradients are scaled to accommodate the variability in tissue
sizes during development. No doubt significant progress in these areas and the
resolution of many other fascinating issues will be forthcoming.
| Box 1. Error correction mechanisms refine tissue patterns established by
morphogen signals A mispositioned cell (red circle) that expresses markers of Domain B (red) is situated in Domain A (green). Three main mechanisms (A-C) have been proposed to correct this type of error.
(A) Selective elimination of mispositioned cells In
Drosophila embryos with one or four copies of bicoid
(bcd), an altered Bcd gradient affects the expression of downstream
genes (Struhl et al., 1989
(B) Sorting of mispositioned cells towards the correct domain
Differential cell adhesion has been implicated in refining and maintaining the
patterns of gene expression (Dahmann and
Basler, 1999
(C) Respecification of mispositioned cells so that they acquire the fate
of their location: the `community effect' Heterotopic transplantations in
the vertebrate hindbrain, for example, indicate that, in contrast to coherent
groups of cells, individual cells are unable to retain their original identity
(Trainor and Krumlauf, 2000
|
|
Note added in proof
A recent study (Rogulja and Irvine,
2005) demonstrates that the slope of a morphogen gradient can
influence cell proliferation in a developing tissue. This supports models of
morphogen action in which the slope of the gradient, in addition to the
concentration of the signal, influences cellular responses, and suggests a
mechanism to coordinate tissue growth with tissue patterning.
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B-like transcription factor Dl. (Right) Graded distribution of
Dl protein; twist and rhomboid are induced by high and low
levels of Dl, respectively. (D) In both Drosophila and
vertebrates, Dpp/BMP signalling operates in a graded manner to pattern several
developing tissues. A Dpp/Screw (Scw) heterodimer activates its heteromeric
complex containing receptor TM serine/threonine kinases. The activated
receptor phosphorylates Mad/Smad transcription factors that, with Med/Smad4
transcription factor, then translocate to the nucleus where they can activate,
in combination with other proteins, target gene expression. In the
Drosophila embryo, high Dpp levels are distributed along the dorsal
midline (top panel), resulting in a peak of phosphorylated Mad (pMad) (middle
panel) and the induction of target genes such as Race (bottom panel).
In the Drosophila embryo, a stepped distribution of Dpp is observed,
resulting in a stepped activation of Mad (see text for details). (E)
Graded Sonic hedgehog (Shh) signalling patterns the ventral neural tube. In
the absence of Shh ligand, the TM protein Patched (Ptch) inhibits Smoothened
(Smo), consequently Gli factors are converted to transcriptional repressors
(GliR). Shh binds to Ptch, relieving repression of Smo, which signals to block
the production of GliR proteins, promoting the generation of Gli activators
(GliA). (Right) A Shh gradient can be visualised in the ventral neural tube
(top panel), which regulates homeodomain protein expression (bottom panel).
(B) Reproduced, with permission, from Ochoa-Espinosa et al.
(Ochoa-Espinosa et al., 2005
